Obligate heterodimer variants of foki cleavage domain

ABSTRACT

Disclosed are methods of making and using engineered FokI cleavage domain variants. Also disclosed are methods, compositions and fusion proteins containing obligate heterodimers of engineered FokI cleavage domain variants and DNA binding domains, such as zinc finger protein (ZFP) domains and transcription activator-like effector (TALE) domains.

The present application is a U.S. National Phase application of andclaims priority from PCT/US2011/045558, filed on 27 Jul. 2011designating the United States, which in turn was based on and claimspriority from U.S. Provisional Patent Application Nos. 61/368,024 filed27 Jul. 2010 all of which applications are incorporated herein byreference.

The research resulting in the invention described herein was supportedin part by funding from the National Institutes of Health GM077291. TheUnited States Government has certain rights in the invention.

BACKGROUND

There have emerged powerful tools, e.g., Zinc finger nucleases (ZFNs)and transcription activator-like effector nucleases (TALENs), fordelivering a targeted genomic double-strand break (DSB) to eitherstimulate local homologous recombination (HR) with investigator-provideddonor DNA or induce gene mutations at the site of cleavage in absence ofa donor by non-homologous end joining (NHEJ), both in plant andmammalian cells, including human cells. ZFNs or TALENs are formed byfusing zinc finger proteins (ZFPs) or transcription activator-likeeffectors (TALEs) to the non-specific cleavage domain of FokIrestriction enzyme. ZFN-mediated (or TALEN-mediated) gene targetingyields high gene modification efficiencies (>10%), in a variety of cellsand cell types by delivering a recombinogenic DSB to the targetedchromosomal locus, using two designed ZFNs (or TALENs). Mechanism of DSBby ZFNs (or TALENs) requires that two ZFN (or TALEN) monomers bind totheir adjacent cognate sites on DNA and the FokI nuclease domainsdimerize to form the active catalytic center for the induction of theDSB. In the case of ZFNs (or TALENs) fused to wild-type FokI cleavagedomains, homodimers may also form, which could limit the efficacy andsafety of the ZFNs (or TALENs) by inducing off-target cleavage. Obligateheterodimer variants of FokI cleavage domain for creating custom ZFNs(or TALENs) are known.

However, there is a need for more efficacy and efficiency of there-engineered obligate heterodimer variants of FokI cleavage domain withminimal cellular toxicity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid and DNA sequence for FokI nuclease domainmutant REL.

FIG. 2 shows the amino acid and DNA sequence for FokI nuclease domainmutant DKK.

FIG. 3 shows the amino acid and DNA sequence for FokI nuclease domainmutant RVEL.

FIG. 4 shows the amino acid and DNA sequence for FokI nuclease domainmutant DKAK.

FIG. 5 shows the amino acid and DNA sequence for FokI nuclease domainvariant with StsI segment containing REL mutations.

FIG. 6 shows the amino acid and DNA sequence for FokI nuclease domainvariant with StsI segment containing DKK mutations.

FIG. 7 shows the amino acid and DNA sequence for FokI wild-type nucleasedomain.

FIG. 8 shows the amino acid and DNA sequence for FokI nuclease domainmutant DA (Cathoman Lab).

FIG. 9 shows the amino acid and DNA sequence for FokI nuclease domainmutant RV (Cathoman Lab).

FIG. 10 shows the amino acid and DNA sequence for FokI nuclease domainmutant EL (Sangamo).

FIG. 11 shows the amino acid and DNA sequence for FokI nuclease domainmutant KK (Sangamo).

FIG. 12 shows the amino acid sequence containing the α4 and α5 helicesthat mediate dimerization between two monomers of the FokI cleavagedomains with the corresponding segment from StsI, an isoschizomer ofFokI endonuclease.

FIG. 13 shows predicted profiles of H-bond interactions between α4helices at the dimer interface of obligate heterodimer variants of FokInuclease domains, based on protein modeling and energy minimization. The3D structure of the FokI dimer was obtained from RCSB Protein Data Bank,which was generated by X-ray diffraction method at a resolution of 2.3A° by Aggarwaal's lab (36). The CCP4 Molecular Graphics software(Version 6.1.2) for Macromolecular X-ray Crystallography was used for 3Dstructure analysis and SPDBV software for protein modeling. Onlyhydrogen bond interactions between α4 helices at the dimer interface areshown. All potential dimer interface interactions (H-bond interactionsand hydrophobic interactions) are listed in Table 2.

FIG. 14 shows the efficiency and efficacy of CCR5 3- and 4-finger ZFNs(generated by fusing the corresponding ZFPs to various obligateheterodimer variants of FokI nuclease domain) using the GFP genetargeting reporter system. HEK293 cells carrying a mutated eGFP reportergene were transiently transfected with a donor plasmid carrying afragment of wild-type GFP and plasmids expressing various 3- or 4-fingerCCR5-specific ZFN constructs, using Lipofectamine 2000 as describedelsewhere (21,27). Transfections of various obligate heterodimervariants and FokI_WT were performed one after another on the same day.After each transfection, the treated cells were split into 2 flasks. GFPpositive cells in about 10,000 treated cells in each flask weredetermined by FACS and then normalized to one million treated cells. Thedifference between the two independent FACS readings is shown as errorbars. A) Frequency of gene correction in HEK293 Flp-In cells of achromosomal mutant GFP reporter disabled by insertion of the CCR5 ZFNtarget sequences using 4-finger ZFNs (28, 29). Quantitative FACSanalyses of the GFP positive cells at 3, 5 and 7 days post-transfectionwith designer CCR5-specific 4-finger ZFNs (constructs carrying theFokI_WT and obligate heterodimer FokI nuclease domain variants) anddonor plasmids. WT: wild type; EL_KK, 4-finger CCR5-ZFNs containing theFokI nuclease domain mutants reported by Miller et al., 2007 (32).RV_DA, 4-finger CCR5-ZFN constructs carrying the FokI nuclease domainmutants reported by Szczepek et al., 2007 (33). REL_DKK, RELV_DKAK andFokI_StsI, 4-finger CCR5-ZFN constructs carrying the FokI nucleasedomain mutants that were generated at PBPL/JHU. B) Top panel: HEK293Flp-In cells 5 days post-transfection as seen in brightfield; Bottompanel: GFP positive cells as seen 5 days post-transfection of HEK293Flp-In cells with 3-finger CCR5-ZFN constructs (carrying Fok_WT, EL_KKor REL_DKK respectively) and donor plasmid. No GFP positive cells wereseen with either donor alone or ZFNs containing FokI_WT nuclease domainsand donor plasmid. C) FACS analyses of the frequency of gene correctionin HEK293 Flp-In cells using 3-finger CCR5 ZFN constructs carryingFokI_WT, EL_KK, REL_DKK or donor alone, respectively. Results from twoindependent transfections, performed on different days, are shown inFigure S1; both transfections showed a similar trend of gene correctionefficiencies for EL_KK and REL_DKK, respectively. The dose responsecurve using titrations of 3-finger ZFN expression plasmids, EL_KK andREL_DKK respectively, at constant donor plasmid, are shown in FIG. 17.D) Analysis of the genotype of nine different individual GFP positiveclones. Five days post-transfection with CCR53-finger ZFNs and the donorplasmids, GFP positive cells were sorted, serially diluted to getindividual clones and grown. The genomic DNA was isolated from the GFPpositive clones and the eGFP gene at the Flp-In locus was PCR amplifiedand digested with BstXI. The mutant eGFP gene has two BstXI sites, wherethe ZFN binding sites are inserted. Correction of the eGFP gene byhomology-directed repair results in the loss of the BstXI sites. The PCRproduct size of the corrected eGFP gene is 930 bp as compared to 990 bpfor the mutant gene. BstXI digestion of the mutant eGFP PCR productgenerates two bands: 450 bp and 540 bp, respectively. Lanes: Control,PCR product of the mutant eGFP gene from untransfected cells before (−)and after (+) digestion with BstXI; GFP⁺1-9, PCR products of 9 differentindividual clones obtained from GFP positive sorted cells before (−) andafter (+) digestion with BstXI; M, 1 Kb ladder. All GFP positive cellsare resistant to BstXI digestion, confirming ZFN-mediated eGFP genecorrection in these cells.

FIG. 15 shows genotypic analysis of the endogenous CCR5 locus at the 3-and 4-finger ZFN target sites of the GFP+ clones *The complete 3- and4-finger ZFN target sites present in hCCR5 gene are highlighted inyellow and blue, respectively, in WT sequences. ^(#)PCR fragmentsamplified from each of the GFP+ clones were subcloned into E. coli. Fourclones from each subcloning experiment were sequenced. The number oftimes the same sequence appeared is shown in brackets. WT denotes wildtype. Insertions are shown in bold lowercase letters. Dots denotedeletions. ^, Homozygous mutations.

FIG. 16 show two independent transfections to confirm the efficiency andefficacy of 3-finger ZFNs (generated by fusing the corresponding ZFPs toEL_KK and REL_DKK variants of FokI nuclease domain, respectively) usingthe GFP gene targeting reporter system. HEK293 cells carrying a mutatedeGFP reporter gene were transiently transfected with a donor plasmidcarrying a fragment of wild-type GFP and the plasmids expressing3-finger CCR5-specific ZFN constructs using Lipofectamine 2000 asdescribed in FIG. 14. For each independent transfection experiment (A &B respectively), the co-transfections of EL_KK and REL_DKK variants wereperformed one after another on the same day. After each co-transfection,the treated cells were split into 2 flasks. GFP positive cells in about10,000 treated cells in each flask were determined by FACS and thennormalized to one million treated cells. The difference between the twoindependent FACS readings is shown as error bars. Independenttransfection experiment shown in FIG. 14C is included as part of thisfigure.

FIG. 17 shows a dose response curve using titrations of 3-finger ZFNexpression plasmids, EL_KK and REL_DKK, respectively. The dose responsecurve was obtained by independent co-transfection of varyingconcentrations (200, 400 and 800 ng respectively) of 3-finger ZFNexpression plasmids at constant donor plasmid concentration (1 μg). FACSanalysis was performed at 3, 5 and 7 days post-transfection as discussedin FIG. 14 and in FIG. 16.

FIG. 18 shows the reduced genome-wide DNA damage levels by REL_DKKobligate heterodimer variant pair of FokI nuclease domain. A),Representative images of cells treated with the DNA cleavage agentetoposide or transfected with the indicated ZFN expression plasmids.Cells were fixed after 30 h and stained with antibodies against 53BP1(red) and then with DAPI (blue). The fraction of cells containing morethan 3 foci is indicated under each panel. The total number of cellsanalyzed is: etoposide, 152; FokI_WT, 245; EL_KK, 282; REL_DKK, 289. B),ZFN expression levels were examined by anti-FokI immunoblot analysis.HEK293 Flp-In cells were transfected with indicated ZFN expressionplasmids and cells were harvested after 30 h. Equal amounts of totalcellular protein was separated by 10% SDS-PAGE gel and transferred toPVDF membrane. The blot was probed with anti-FokI antibody. The ZFNsmigrate as a single band on the gel. Western blot analysis showscomparable levels ZFN expression for various obligate heterodimervariants in HEK293 Flp-In cells.

DESCRIPTION

Disclosed herein are polypeptides comprising an engineered FokI cleavagedomain variant comprising a mutation in at least three or more wild-typeamino acid residues, the engineered FokI cleavage domain variant formingan obligate heterodimer with a second engineered FokI cleavage domainvariant with at least one different mutation in one or more wild-typeamino acid residues.

In particular embodiments, disclosed are fusion polypeptides containingthe engineered FokI cleavage domain variants useful for targetedcleavage of cellular chromatin and for targeted alteration of a cellularnucleotide sequence, e.g., by targeted cleavage followed bynon-homologous end joining or by targeted cleavage followed byhomologous recombination between an exogenous polynucleotide (comprisingone or more regions of homology with the cellular nucleotide sequence)and a genomic sequence.

Exemplary engineered FokI cleavage domain variant are shown in FIGS.1-6. The variants in include at least three mutations such that theyform heterodimers with each other, but not homodimers. This increasesthe specificity of DNA cleavage and/or increases the concentration ofthe intended complex (by reducing or eliminating competition fromhomodimers). When incorporated into fusion proteins, such as zinc fingernucleases (ZFN) and transcription activator-like effector nucleases(TALENs), these variants induce gene modification at the intended target(both at an endogenous locus and when tested using an integrated GFPreporter assay) while significantly reducing genome-wide off-target DNAcleavage as compared to wild-type FokI cleavage domain variant.

Thus, the engineered FokI cleavage domain variant described hereinsignificantly impair homodimer function, since forcing two copies of thesame variant to interact reduces or abolishes gene modification. Reducedhomodimer function provides improved ZFN or TALEN cleavage specificityin vivo, without any decrease in either ZFN or TALEN expression or theability to stimulate modification of the desired target site.

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K.

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins or TALE have DNA-binding, RNA-binding and protein-bindingactivity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP. Zinc finger bindingdomains can be “engineered” to bind to a predetermined nucleotidesequence. Non-limiting examples of methods for engineering zinc fingerproteins are design and selection. A designed zinc finger protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPdesigns and binding data.

A “selected” zinc finger protein is a protein not found in nature whoseproduction results primarily from an empirical process such as phagedisplay, interaction trap or hybrid selection.

Examples of Designed Zinc Finger Nucleases can be found in US PublishedPatent Application No. 2010/0055793.

Transcription activator-like effector (TALE) protein (or binding domain)is a protein, or a domain within a larger protein, that binds DNA in asequence-specific manner. Transcription activator-like effector (TALE)domains can be “engineered” to bind to a predetermined nucleotidesequence. Examples of TALE proteins and domains can be found in Milleret al., A TALE nuclease architecture for efficient genome editing, Nat.Biotechnol. 2011 February; 29(2):143-8. Epub 2010 Dec. 22; andHockemeyer et al., Genetic engineering of human pluripotent cells usingTALE nucleases, Nat. Biotechnol. 2011 Jul. 7. doi: 10.1038/nbt.1927.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value there between or there above), preferablybetween about 100 and 1,000 nucleotides in length (or any integer therebetween), more preferably between about 200 and 500 nucleotides inlength.

A “homologous, non-identical sequence” refers to a first sequence whichshares a degree of sequence identity with a second sequence, but whosesequence is not identical to that of the second sequence. For example, apolynucleotide comprising the wild-type sequence of a mutant gene ishomologous and non-identical to the sequence of the mutant gene. Incertain embodiments, the degree of homology between the two sequences issufficient to allow homologous recombination there between, utilizingnormal cellular mechanisms. Two homologous non-identical sequences canbe any length and their degree of non-homology can be as small as asingle nucleotide (e.g., for correction of a genomic point mutation bytargeted homologous recombination) or as large as 10 or more kilobases(e.g., for insertion of a gene at a predetermined ectopic site in achromosome). Two polynucleotides comprising the homologous non-identicalsequences need not be the same length. For example, an exogenouspolynucleotide (i.e., donor polynucleotide) of between 20 and 10,000nucleotides or nucleotide pairs can be used.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. An approximatealignment for nucleic acid sequences is provided by the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics2:482-489 (1981). This algorithm can be applied to amino acid sequencesby using the scoring matrix developed by Dayhoff, Atlas of ProteinSequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, NationalBiomedical Research Foundation, Washington, D.C., USA, and normalized byGribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplaryimplementation of this algorithm to determine percent identity of asequence is provided by the Genetics Computer Group (Madison, Wis.) inthe “BestFit” utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.). A preferred method of establishing percent identity inthe context of the present disclosure is to use the MPSRCH package ofprograms copyrighted by the University of Edinburgh, developed by JohnF. Collins and Shane S. Sturrok, and distributed by IntelliGenetics,Inc. (Mountain View, Calif.). From this suite of packages theSmith-Waterman algorithm can be employed where default parameters areused for the scoring table (for example, gap open penalty of 12, gapextension penalty of one, and a gap of six). From the data generated the“Match” value reflects sequence identity. Other suitable programs forcalculating the percent identity or similarity between sequences aregenerally known in the art, for example, another alignment program isBLAST, used with default parameters. For example, BLASTN and BLASTP canbe used using the following default parameters: genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST. Withrespect to sequences described herein, the range of desired degrees ofsequence identity is approximately 80% to 100% and any integer valuetherebetween. Typically the percent identities between sequences are atleast 70-75%, preferably 80-82%, more preferably 85-90%, even morepreferably 92%, still more preferably 95%, and most preferably 98%sequence identity.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat allow formation of stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two nucleic acid, or twopolypeptide sequences are substantially homologous to each other whenthe sequences exhibit at least about 70%-75%, preferably 80%-82%, morepreferably 85%-90%, even more preferably 92%, still more preferably 95%,and most preferably 98% sequence identity over a defined length of themolecules, as determined using the methods above. As used herein,substantially homologous also refers to sequences showing completeidentity to a specified DNA or polypeptide sequence. DNA sequences thatare substantially homologous can be identified in a Southernhybridization experiment under, for example, stringent conditions, asdefined for that particular system. Defining appropriate hybridizationconditions is within the skill of the art. See, e.g., Sambrook et al.,supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D.Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

Selective hybridization of two nucleic acid fragments can be determinedas follows. The degree of sequence identity between two nucleic acidmolecules affects the efficiency and strength of hybridization eventsbetween such molecules. A partially identical nucleic acid sequence willat least partially inhibit the hybridization of a completely identicalsequence to a target molecule Inhibition of hybridization of thecompletely identical sequence can be assessed using hybridization assaysthat are well known in the art (e.g., Southern (DNA) blot, Northern(RNA) blot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a reference nucleic acidsequence, and then by selection of appropriate conditions the probe andthe reference sequence selectively hybridize, or bind, to each other toform a duplex molecule. A nucleic acid molecule that is capable ofhybridizing selectively to a reference sequence under moderatelystringent hybridization conditions typically hybridizes under conditionsthat allow detection of a target nucleic acid sequence of at least about10-14 nucleotides in length having at least approximately 70% sequenceidentity with the sequence of the selected nucleic acid probe. Stringenthybridization conditions typically allow detection of target nucleicacid sequences of at least about 10-14 nucleotides in length having asequence identity of greater than about 90-95% with the sequence of theselected nucleic acid probe. Hybridization conditions useful forprobe/reference sequence hybridization, where the probe and referencesequence have a specific degree of sequence identity, can be determinedas is known in the art (see, for example, Nucleic Acid Hybridization: APractical Approach, editors B. D. Hames and S. J. Higgins, (1985)Oxford; Washington, D.C.; IRL Press).

Conditions for hybridization are well-known to those of skill in theart. Hybridization stringency refers to the degree to whichhybridization conditions disfavor the formation of hybrids containingmismatched nucleotides, with higher stringency correlated with a lowertolerance for mismatched hybrids. Factors that affect the stringency ofhybridization are well-known to those of skill in the art and include,but are not limited to, temperature, pH, ionic strength, andconcentration of organic solvents such as, for example, formamide anddimethylsulfoxide. As is known to those of skill in the art,hybridization stringency is increased by higher temperatures, lowerionic strength and lower solvent concentrations.

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of the sequences, base composition of thevarious sequences, concentrations of salts and other hybridizationsolution components, the presence or absence of blocking agents in thehybridization solutions (e.g., dextran sulfate, and polyethyleneglycol), hybridization reaction temperature and time parameters, as wellas, varying wash conditions. The selection of a particular set ofhybridization conditions is selected following standard methods in theart (see, for example, Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, (1989) Cold Spring Harbor, N.Y.).

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and is variouslyknown as “non-crossover gene conversion” or “short tract geneconversion,” because it leads to the transfer of genetic informationfrom the donor to the target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

An “accessible region” is a site in cellular chromatin in which a targetsite present in the nucleic acid can be bound by an exogenous moleculewhich recognizes the target site. Without wishing to be bound by anyparticular theory, it is believed that an accessible region is one thatis not packaged into a nucleosomal structure. The distinct structure ofan accessible region can often be detected by its sensitivity tochemical and enzymatic probes, for example, nucleases.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylates,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “FokI cleavage domain variant” is a polypeptide sequence which can, inconjunction with a second polypeptide (either identical or different)form a complex having cleavage activity (preferably double-strandcleavage activity). The terms “first and second FokI cleavage domainvariant;” “+ and − FokI cleavage domain variant” and “right and leftFokI cleavage domain variant” are used interchangeably to refer to pairsof FokI cleavage domain variant that dimerize. In particularembodiments, the FokI cleavage domain variant may be referred to as ahalf-domain FokI cleavage variant. FokI restriction endonucleases havebeen described in U.S. Pat. Nos. 5,356,802, 5,436,150, 5,487,994,5,792,640, 5,916,794, and 6,265,196.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and a cleavage domain) and fusion nucleicacids (for example, a nucleic acid encoding the fusion protein describedsupra). Examples of the second type of fusion molecule include, but arenot limited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of a mRNA. Gene products also include RNAs whichare modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression.

“Eucaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFP or TALEDNA-binding domain is fused to a cleavage domain, the ZFP or the TALEDNA-binding domain and the cleavage domain are in operative linkage if,in the fusion polypeptide, the ZFP or TALE DNA-binding domain portion isable to bind its target site and/or its binding site, while the cleavagedomain is able to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246.

An “engineered FokI cleavage domain variant” is a FokI cleavage domainthat has been modified so as to form obligate heterodimers with anotherFokI cleavage domain variant (e.g., another engineered FokI cleavagedomain variant). Engineered FokI cleavage domain variant (also bereferred to as dimerization domain mutants) minimize or preventhomodimerization. Amino acid residues at positions 446, 447, 479, 483,484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 ofFokI all can be targets for influencing dimerization of the FokIcleavage domain variants. Numbering of amino acid residues in the FokIprotein is according to Wah et al., (1998) Proc Natl Acad Sci USA95:10564-10569.

Described herein are engineered FokI cleavage domain variants thatcontain a mutation in at least three or more wild-type amino acidresidues and that form an obligate heterodimer. Exemplary mutant FokIcleavage domain variants are shown in FIGS. 1-6. In certain embodiments,the FokI cleavage domain variant includes mutations in at least threeamino acid residues at positions 483, 486, 487, 490, 499 or 538 ofwild-type FokI, see FIGS. 1-4 and Examples.

Specifically, the engineered FokI cleavage domain variants, describedherein, were prepared by mutating certain positions in a wild-type FokIcleavage domain sequence to produce an engineered FokI cleavage domainvariant.

Examples of the engineered FokI cleavage domain variant, may include thepolypeptide designated D483R:Q486E:I499L (SEQ ID NO: 1), the polypeptidedesignated R487D:E490K:I538K (SEQ ID NO: 3), the polypeptide designatedD483R:Q486E:I499L:I538V (SEQ ID NO: 5), or the polypeptide designatedR487D:E490K:I499A:I538K (SEQ ID NO: 7).

In further embodiments, the engineered FokI cleavage domain variant mayinclude the polypeptide designated:

(SEQ ID NO: 66) LDSKAYSEGFPLTASHT

AM

RYLRQFTERKEE L KPTWWDIAPEHLDN TYFAYVSGSSFSGNYKEQLQKFRQDT,or the polypeptide designated: (SEQ ID NO: 67) ILDSKAYSEGFPLTASHTDAMG

YL

QFTERKEEIKPTWWDIAPEHLD NTYFAYVSGSFSGNYKEQLQKFRQ

T.

In certain aspects, the segment from the wild-type FokI cleavage domainvariant,

(SEQ ID NO: 23) IVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITN,is replaced with the polypeptide designated: (SEQ ID NO: 66)LDSKAYSEGFPLTASHT

AM

RYLRQFTERKEE L KPTWWDIAPEHLDN TYFAYVSGSSFSGNYKEQLQKFRQDT,or the polypeptide designated: (SEQ ID NO: 67) ILDSKAYSEGFPLTASHTDAMG

YL

QFTERKEEIKPTWWDIAPEHLD NTYFAYVSGSFSGNYKEQLQKFRQ

T.

Engineered FokI cleavage domain variants described herein can beprepared using any suitable method, for example, by site-directedmutagenesis of wild-type FokI cleavage domain variant (FokI) asdescribed in the examples. The engineered FokI cleavage domain variantsdescribed herein are obligate heterodimer mutants in which aberrantcleavage is minimized or abolished.

In yet other embodiments, disclosed are heterodimers made up of a firstengineered FokI cleavage domain variant including a mutation in at leastthree or more wild-type amino acid residues, and a second differentengineered FokI cleavage domain variant with at least one differentmutation in one or more wild-type amino acid residues. In certainembodiments, the mutations may be in at least three amino acid residuesat positions 483, 486, 487, 490, 499 or 538 of wild-type FokI. Incertain aspects the heterodimers are made up of a first engineered FokIcleavage domain variant including a mutation in at least three or morewild-type amino acid residues, and a second different engineered FokIcleavage domain variant with different mutations in three or morewild-type amino acid residues.

In certain embodiments, the heterodimer may include a first engineeredFokI cleavage domain variant that is different from the secondengineered FokI cleavage domain variant, and both independently containa polypeptide selected from the polypeptide designated D483R:Q486E:I499L(SEQ ID NO: 1), the polypeptide designated R487D:E490K:I538K (SEQ ID NO:3), the polypeptide designated D483R:Q486E:I499L:I538V (SEQ ID NO: 5) orthe polypeptide designated R487D:E490K:I499A:I538K (SEQ ID NO: 7).

In an exemplary embodiment, the heterodimer is made up of an engineeredFokI cleavage domain variant containing the polypeptide designatedD483R:Q486E:I499L (SEQ ID NO: 1), and the other containing thepolypeptide designated R487D:E490K:I538K (SEQ ID NO: 3).

In certain aspects, the obligate heterodimer may include a first monomercontaining the polypeptide designated D483R:Q486E:I499L (SEQ ID NO: 1),and a second monomer containing the polypeptide designatedR487D:E490K:I538K (SEQ ID NO: 3). In yet other aspects, the obligateheterodimer may include a first monomer containing the polypeptidedesignated D483R:Q486E:I499L:I538V (SEQ ID NO: 5), and a second monomercontaining the polypeptide designated R487D:E490K:I499A:I538K (SEQ IDNO: 7).

In yet other aspects, the obligate heterodimer may include a firstmonomer containing the polypeptide designated:

(SEQ ID NO: 66) LDSKAYSEGFPLTASHT

AM

RYLRQFTERKEE L KPTWWDIAPEHLDN TYFAYVSGSSFSGNYKEQLQKFRQDT,and a second monomer containing the polypeptide designated:(SEQ ID NO: 67) LDSKAYSEGFPLTASHTDAMG

YL

QFTERKEEIKPTWWDIAPEHLDN TYFAYVSGSFSGNYKEQLQKFRQ

T.

In other embodiments, the engineered FokI cleavage domain variantdescribed herein are advantageously used in fusion proteins with aDNA-binding domain, such as zinc finger proteins (ZFPs) or transcriptionactivator-like effector (TALE) proteins, to specifically target sitesfor cleavage in any cell.

In certain embodiments, the fusion protein may include a DNA-bindingdomain and at least one engineered FokI cleavage domain variantcomprising a mutation in at least three or more wild-type amino acidresidues, the engineered FokI cleavage domain variant forming anobligate heterodimer with a second engineered FokI cleavage domainvariant with at least one different mutation in one or more wild-typeamino acid residues. The DNA-binding domain may include zinc fingerprotein (ZFP) or transcription activator-like effector (TALE) proteindomains.

In further embodiments, disclosed are methods for making and using theengineered FokI cleavage domain variants, the obligate heterodimercontaining two FokI cleavage domain variants, and the fusion proteincontaining the obligate heterodimer containing two FokI cleavage domainvariants and DNA binding domains containing, e.g., ZFPs or TALEs, asdescribed below.

ZFNs or TALENs and methods for design and construction of fusionproteins (and polynucleotides encoding same) are known to those of skillin the art. The ZFNs or TALEs described herein may be delivered to atarget cell by any suitable means. Methods of delivering proteinscomprising ZFNs or TALENs are described. ZFNs or TALENs as describedherein may also be delivered using vectors containing sequences encodingone or more ZFNs or TALENs. Any vector systems may be used including,but not limited to, plasmid vectors, retroviral vectors, lentiviralvectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors andadeno-associated virus vectors, etc.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding ZFNs or TALENs comprising engineeredcleavage domains in cells (e.g., mammalian cells) and target tissues.Such methods can also be used to administer such nucleic acids to cellsin vitro. In certain embodiments, nucleic acids encoding ZFNs or TALENsare administered for in vivo or ex vivo gene therapy uses. Non-viralvector delivery systems include DNA plasmids, naked nucleic acid, andnucleic acid complexed with a delivery vehicle such as a liposome orpoloxamer. Viral vector delivery systems include DNA and RNA viruses,which have either episomal or integrated genomes after delivery to thecell. For a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993);Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiologyand Immunology Doerfler and Bohm (eds.) (1995); and Yu et al., GeneTherapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids encoding ZFNs or TALENsinclude electroporation, lipofection, microinjection, biolistics,virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acidconjugates, naked DNA, artificial virions, and agent-enhanced uptake ofDNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) canalso be used for delivery of nucleic acids. Additional exemplary nucleicacid delivery systems include those provided by Amaxa Biosystems(Cologne, Germany), Maxcyte, Inc. (Rockville, Md.) and BTX MolecularDelivery Systems (Holliston, Mass.). Lipofection and lipofectionreagents are sold commercially (e.g., Transfectam™ and Lipofectin™).Cationic and neutral lipids that are suitable for efficientreceptor-recognition lipofection of polynucleotides include those ofFeigner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivoadministration) or target tissues (in vivo administration).

The preparation of lipid nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); and Ahmad et al., Cancer Res.52:4817-4820 (1992).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding ZFNs or TALENs comprising engineered FokI cleavage domainvariant as described herein take advantage of highly evolved processesfor targeting a virus to specific cells in the body and trafficking theviral payload to the nucleus. Viral vectors can be administered directlyto patients (in vivo) or they can be used to treat cells in vitro andthe modified cells are administered to patients (ex vivo). Conventionalviral based systems for the delivery of ZFNs or TALENs include, but arenot limited to, retroviral, lentivirus, adenoviral, adeno-associated,vaccinia and herpes simplex virus vectors for gene transfer. Integrationin the host genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/U594/05700).

In applications in which transient expression of a pair of ZFN or TALENfusion proteins is preferred, adenoviral based systems can be used.Adenoviral based vectors are capable of very high transductionefficiency in many cell types and do not require cell division. Withsuch vectors, high titer and high levels of expression have beenobtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

In certain embodiments, the vector is an adenovirus vector. Thus,described herein are adenovirus (Ad) vectors for introducingheterologous sequences (e.g., ZFNs or TALENs) into cells.

Non-limiting examples of Ad vectors that can be used in the presentapplication include recombinant (such as E1-deleted), conditionallyreplication competent (such as oncolytic) and/or replication competentAd vectors derived from human or non-human serotypes (e.g., Ad5, Ad11,Ad35, or porcine adenovirus-3); and/or chimeric Ad vectors (such asAd5/35) or tropism-altered Ad vectors with engineered fiber (e.g., knobor shaft) proteins (such as peptide insertions within the HI loop of theknob protein). Also useful are “gutless” Ad vectors, e.g., an Ad vectorin which all adenovirus genes have been removed, to reduceimmunogenicity and to increase the size of the DNA payload. This allows,for example, simultaneous delivery of sequences encoding ZFNs or TALENsand a donor sequence. Such gutless vectors are especially useful whenthe donor sequences include large transgenes to be integrated viatargeted integration.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer, and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in cells that provide one or more of thedeleted gene functions in trans. For example, human 293 cells supply E1function. Ad vectors can transduce multiple types of tissues in vivo,including non-dividing, differentiated cells such as those found inliver, kidney and muscle. Conventional Ad vectors have a large carryingcapacity. An example of the use of an Ad vector in a clinical trialinvolved polynucleotide therapy for antitumor immunization withintramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-1089(1998)).

Additional examples of the use of adenovirus vectors for gene transferin clinical trials include Rosenecker et al., Infection 24:1 5-10(1996); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al.,Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513(1998).

In certain embodiments, the Ad vector is a chimeric adenovirus vector,containing sequences from two or more different adenovirus genomes. Forexample, the Ad vector can be an Ad5/35 vector. Ad5/35 is created byreplacing one or more of the fiber protein genes (knob, shaft, tail,penton) of Ad5 with the corresponding fiber protein gene from a B groupadenovirus such as, for example, Ad35. The Ad5/35 vector andcharacteristics of this vector are described, for example, in Ni et al.(2005) “Evaluation of biodistribution and safety of adenovirus vectorscontaining group B fibers after intravenous injection into baboons,” HumGene Ther 16:664-677; Nilsson et al. (2004) “Functionally distinctsubpopulations of cord blood CD34+ cells are transduced by adenoviralvectors with serotype 5 or 35 tropism,” Mol Ther 9:377-388; Nilsson etal. (2004) “Development of an adenoviral vector system with adenovirusserotype 35 tropism; efficient transient gene transfer into primarymalignant hematopoietic cells,” J Gene Med 6:631-641; Schroers et al.(2004) “Gene transfer into human T lymphocytes and natural killer cellsby Ad5/F35 chimeric adenoviral vectors,” Exp Hematol 32:536-546;Seshidhar et al. (2003) “Development of adenovirus serotype 35 as a genetransfer vector,” Virology 311:384-393; Shayakhmetov et al. (2000)“Efficient gene transfer into human CD34(+) cells by a retargetedadenovirus vector,” J Virol 74:2567-2583; and Soya et al. (2004), “Atumor-targeted and conditionally replicating oncolytic adenovirus vectorexpressing TRAIL for treatment of liver metastases,” Mol Ther 9:496-509.

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a ZFNs orTALENs nucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1(granulocytes), and lad (differentiated antigen presenting cells) (seeInaba et al., J. Exp. Med. 176:1693-1702 (1992)).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFNs or TALENs nucleic acids can also be administereddirectly to an organism for transduction of cells in vivo.Alternatively, naked DNA can be administered. Administration is by anyof the routes normally used for introducing a molecule into ultimatecontact with blood or tissue cells including, but not limited to,injection, infusion, topical application and electroporation. Suitablemethods of administering such nucleic acids are available and well knownto those of skill in the art, and, although more than one route can beused to administer a particular composition, a particular route canoften provide a more immediate and more effective reaction than anotherroute.

Methods for introduction of DNA into hematopoietic stem cells are knownand Vectors useful for introduction of transgenes into hematopoieticstem cells, e.g., CD34+ cells, include adenovirus Type 35. Vectorssuitable for introduction of transgenes into immune cells (e.g.,T-cells) include non-integrating lentivirus vectors. See, for example,Ory et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

As noted above, the disclosed methods and compositions can be used inany type of cell including, but not limited to, prokaryotic cells,fungal cells, Archaeal cells, plant cells, insect cells, animal cells,vertebrate cells, mammalian cells and human cells. Suitable cell linesfor protein expression are known to those of skill in the art andinclude, but are not limited to COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44,CHO-DUXB11), VERO, MDCK, W138, V79, B14AF28-G3, BHK, HaK, NS0,SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6,insect cells such as Spodoptera fugiperda (Sf), and fungal cells such asSaccharomyces, Pischia and Schizosaccharomyces. Progeny, variants andderivatives of these cell lines can also be used.

The disclosed cleavage domains are advantageously used in combinationwith zinc finger proteins or transcription activator-like effector(TALE) proteins to cleave DNA and minimize off-target site cleavage (ascompared to ZFNs or TALENs comprising wild-type or homodimerizingcleavage domains). Cleavage can be at a region of interest in cellularchromatin (e.g., at a desired or predetermined site in a genome, forexample, in a gene, either mutant or wild-type); to replace a genomicsequence (e.g., a region of interest in cellular chromatin) with ahomologous non-identical sequence (i.e., targeted recombination); todelete a genomic sequence by cleaving DNA at one or more sites in thegenome, which cleavage sites are then joined by non-homologous endjoining (NHEJ); to screen for cellular factors that facilitatehomologous recombination; and/or to replace a wild-type sequence with amutant sequence, or to convert one allele to a different allele.

Accordingly, the disclosed engineered FokI cleavage domain variant canbe used in any ZFNs or TALENs for any method in which specificallytargeted cleavage is desirable and/or to replace any genomic sequencewith a homologous, non-identical sequence. For example, a mutant genomicsequence can be replaced by its wild-type counterpart, thereby providingmethods for treatment of e.g., genetic disease, inherited disorders,cancer, and autoimmune disease. In like fashion, one allele of a genecan be replaced by a different allele using the methods of targetedrecombination disclosed herein. Indeed, any pathology dependent upon aparticular genomic sequence, in any fashion, can be corrected oralleviated using the methods and compositions disclosed herein.

Exemplary genetic diseases include, but are not limited to,achondroplasia, achromatopsia, acid maltase deficiency, adenosinedeaminase deficiency (OMIM No. 102700), adrenoleukodystrophy, aicardisyndrome, alpha-1 antitrypsin deficiency, alpha-thalassemia, androgeninsensitivity syndrome, apert syndrome, arrhythmogenic rightventricular, dysplasia, ataxia telangictasia, barth syndrome,beta-thalassemia, blue rubber bleb nevus syndrome, canavan disease,chronic granulomatous diseases (CGD), cri du chat syndrome, cysticfibrosis, dercum's disease, ectodermal dysplasia, fanconi anemia,fibrodysplasia ossificans progressive, fragile X syndrome, galactosemis,Gaucher's disease, generalized gangliosidoses (e.g., GM1),hemochromatosis, the hemoglobin C mutation in the 6.sup.th codon ofbeta-globin (HbC), hemophilia, Huntington's disease, Hurler Syndrome,hypophosphatasia, Klinefleter syndrome, Krabbes Disease, Langer-GiedionSyndrome, leukocyte adhesion deficiency (LAD, OMIM No. 116920),leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome,mucopolysaccharidosis (MPS), nail patella syndrome, nephrogenic diabetesinsipdius, neurofibromatosis, Neimann-Pick disease, osteogenesisimperfecta, porphyria, Prader-Willi syndrome, progeria, Proteussyndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome,Sanfilippo syndrome, severe combined immunodeficiency (SCID), Shwachmansyndrome, sickle cell disease (sickle cell anemia), Smith-Magenissyndrome, Stickler syndrome, Tay-Sachs disease, Thrombocytopenia AbsentRadius (TAR) syndrome, Treacher Collins syndrome, trisomy, tuberoussclerosis, Turner's syndrome, urea cycle disorder, von Hippel-Landaudisease, Waardenburg syndrome, Williams syndrome, Wilson's disease,Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome (XLP,OMIM No. 308240).

Additional exemplary diseases that can be treated by targeted DNAcleavage and/or homologous recombination include acquiredimmunodeficiencies, lysosomal storage diseases (e.g., Gaucher's disease,GM1, Fabry disease and Tay-Sachs disease), mucopolysaccahidosis (e.g.Hunter's disease, Hurler's disease), hemoglobinopathies (e.g., sicklecell diseases, HbC, α-thalassemia, β-thalassemia) and hemophilias.

Such methods also allow for treatment of infections (viral or bacterial)in a host (e.g., by blocking expression of viral or bacterial receptors,thereby preventing infection and/or spread in a host organism); to treatgenetic diseases.

Targeted cleavage of infecting or integrated viral genomes can be usedto treat viral infections in a host. Additionally, targeted cleavage ofgenes encoding receptors for viruses can be used to block expression ofsuch receptors, thereby preventing viral infection and/or viral spreadin a host organism. Targeted mutagenesis of genes encoding viralreceptors (e.g., the CCR5 and CXCR4 receptors for HIV) can be used torender the receptors unable to bind to virus, thereby preventing newinfection and blocking the spread of existing infections.

Non-limiting examples of viruses or viral receptors that may be targetedinclude herpes simplex virus (HSV), such as HSV-1 and HSV-2, varicellazoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV),HHV6 and HHV7. The hepatitis family of viruses includes hepatitis Avirus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the deltahepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus(HGV). Other viruses or their receptors may be targeted, including, butnot limited to, Picornaviridae (e.g., polioviruses, etc.);Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.);Flaviviridae; Coronaviridae; Reoviridae; Bimaviridae; Rhabodoviridae(e.g., rabies virus, etc.); Filoviridae; Paramyxoviridae (e.g., mumpsvirus, measles virus, respiratory syncytial virus, etc.);Orthomyxoviridae (e.g., influenza virus types A, B and C, etc.);Bunyaviridae; Arenaviridae; Retroviradae; lentiviruses (e.g., HTLV-I;HTLV-II; HIV-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.) HIV-II);simian immunodeficiency virus (SIV), human papillomavirus (HPV),influenza virus and the tick-borne encephalitis viruses. See, e.g.Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2ndEdition (B. N. Fields and D. M. Knipe, eds. 1991), for a description ofthese and other viruses. Receptors for HIV, for example, include CCR5and CXCR-4.

Among the genes which can be cleaved is CCR5 co-receptor (hCCR5) throughwhich HIV gains entry into cells early in the infection. Thus, in oneaspect, described herein are compositions and methods useful fordisrupting the CCR5 gene in cells comprising an engineered fusionprotein including zinc finger binding domain or transcriptionactivator-like effector (TALE) domain to bind to a CCR5 target sequenceand an engineered FokI cleavage domain variant, wherein said fusionprotein binds to the CCR5 gene and cleaves the CCR5 gene. The mutationcan be associated with any function of CCR5, e.g. the ability of an HIVvirus to enter a host cell via the CCR5 co-receptor. CCR5 genes can bedisrupted for a variety of purposes. For example, after cleavage ofCCR5, the gene can be repaired by non-homologous end joining in the cellto give rise to a CCR5 gene mutation that inactivates the CCR5 receptorto produce HIV resistant cells. Alternatively, CCR5 receptor can bedisrupted by replacing a wild type sequence with a CCR5Δ32 mutation. Inone embodiment, a CCR5 chromosomal gene locus can serve as a “safeharbor” for the introduction of transgenes. That is, functions of CCR5may be expendable, so that the gene can be cleaved and one of moretherapeutic transgenes of interest can be inserted at the cleavage sitefor functional complementation in cells. In one embodiment, the CCR5gene is a human gene, where one or more genes of interest can beintroduced and expressed ectopically. These genes can be marker genes(e.g. neomycin or green fluorescent protein (GFP)) or genes applicablefor human therapeutics.

In an exemplary embodiment, the fusion protein includes 3- or 4-zincfinger proteins (ZFP's) that target CCR5 of human cells, and theobligate heterodimer comprises a first monomer containing thepolypeptide designated D483R:Q486E:I499L, and a second monomercontaining the polypeptide designated R487D:E490K:I538K.

In particular embodiments, the zinc finger proteins (ZFPs) may includeZF1, ZF2, ZF3, ZF4, ZF5 or ZF6, described in Table 1.

TABLE 1 ZF designs for the chosen targets within various mammalian genesTriplet DNA coding sequence/contact residues ZFN target site subsites −1 to +6 positions) of the α-helix  Gene 5′-3′ 5′-3′ for the ZF designshCCR5 GCT GCC GCC c ZF1 GCC c GAA CGC GGA ACG CTG GCC CGC(SEQ ID NO: 52) (SEQ ID NO: 53) E R G T L A R (SEQ ID NO: 54) ZF2 GCC gGAC CGC TCG GAC TTG ACG CGC (SEQ ID NO: 55) D R S D L T R(SEQ ID NO: 56) ZF3 GCT g CAA TCC TCT GAC TTG ACG CGC (SEQ ID NO: 57)Q S S D L T R (SEQ ID NO: 58) GAA GGG GAC a ZF4 GAC aGAC AGA TCC AAC CTT ACC CGC (SEQ ID NO: 59) (SEQ ID NO: 60) D R S NL T R(SEQ ID NO: 61) ZF5 GGG g CGC AGC GAT CAT CTC ACC AAA (SEQ ID NO: 62)R S D H L T K (SEQ ID NO: 63) ZF6 GAA g CAA TCC TCT AAT CTC GCT CGC(SEQ ID NO: 64) Q S S N L A R (SEQ ID NO: 65)

In other embodiments, disclosed is a method of cleaving a gene ofinterest in a cell, the method comprising: providing a fusion proteincomprising a DNA binding domain, e.g., zinc finger (ZF) binding domainor transcription activator-like effector (TALE) domain and an engineeredFokI cleavage domain variant, wherein the zinc finger binding domain ortranscription activator-like effector (TALE) domain binds to a targetsite in the gene of interest; and contacting the cell with the fusionprotein under conditions such that the gene of interest is cleaved. Inyet another embodiment, a composition is disclosed useful for disruptinga CCR5 gene in a cell, comprising an engineered fusion protein whichcomprises zinc finger (ZF) binding domain or transcriptionactivator-like effector (TALE) domain to bind a gene of interest and anengineered FokI cleavage domain variant, wherein the fusion proteinbinds to and cleaves the gene of interest.

In particular embodiments, the gene of interest is CCR5, the zinc fingerbinding domain binds to a target site in the CCR5 gene, and the CCR5gene is cleaved. In yet other aspects, the zinc finger binding domaincomprises, as a recognition region, one of the six sequences shown forhCCR5 in Table 1. In yet another aspect, the recognition region of eachof the three zinc fingers is ZF1, ZF2 or ZF3, or ZF4, ZF5 or ZF6.

Heterodimeric cleavage domain variants as described herein provide broadutility for improving ZFN or TALEN specificity in gene modificationapplications. These variant cleavage domains may be readily incorporatedinto any existing ZFN or TALEN by either site directed mutagenesis orsubcloning to improve the in vivo specificity of any ZFN or TALENdimers.

As noted above, the compositions and methods described herein can beused for gene modification, gene correction, and gene disruption.Non-limiting examples of gene modification includes homology directedrepair (HDR)-based targeted integration; HDR-based gene correction;HDR-based gene modification; HDR-based gene disruption; NHEJ-based genedisruption and/or combinations of HDR, NHEJ, and/or single strandannealing (SSA). Single-Strand Annealing (SSA) refers to the repair of adouble strand break between two repeated sequences that occur in thesame orientation by resection of the DSB by 5′-3′ exonucleases to exposethe 2 complementary regions. The single-strands encoding the 2 directrepeats then anneal to each other, and the annealed intermediate can beprocessed such that the single-stranded tails (the portion of thesingle-stranded DNA that is not annealed to any sequence) are bedigested away, the gaps filled in by DNA Polymerase, and the DNA endsrejoined. This results in the deletion of sequences located between thedirect repeats.

Compositions comprising cleavage domains (e.g., ZFNs or TALENs) andmethods described herein can also be used in the treatment of variousgenetic diseases and/or infectious diseases.

The compositions and methods can also be applied to stem cell basedtherapies, including but not limited to: (a) Correction of somatic cellmutations by short patch gene conversion or targeted integration formonogenic gene therapy; (b) Disruption of dominant negative alleles; (c)Disruption of genes required for the entry or productive infection ofpathogens into cells; (d) Enhanced tissue engineering, for example, by:(i) Modifying gene activity to promote the differentiation or formationof functional tissues, and/or (ii) Disrupting gene activity to promotethe differentiation or formation of functional tissues; (e) Blocking orinducing differentiation, for example, by: (i) Disrupting genes thatblock differentiation to promote stem cells to differentiate down aspecific lineage pathway, (ii) Targeted insertion of a gene or siRNAexpression cassette that can stimulate stem cell differentiation, (iii)Targeted insertion of a gene or siRNA expression cassette that can blockstem cell differentiation and allow better expansion and maintenance ofpluripotency, and/or (iv) Targeted insertion of a reporter gene in framewith an endogenous gene that is a marker of pluripotency ordifferentiation state that would allow an easy marker to scoredifferentiation state of stem cells and how changes in media, cytokines,growth conditions, expression of genes, expression of siRNA molecules,exposure to antibodies to cell surface markers, or drugs alter thisstate; (f) Somatic cell nuclear transfer, for example, a patient's ownsomatic cells can be isolated, the intended target gene modified in theappropriate manner, cell clones generated (and quality controlled toensure genome safety), and the nuclei from these cells isolated andtransferred into unfertilized eggs to generate patient-specific hEScells that could be directly injected or differentiated beforeengrafting into the patient, thereby reducing or eliminating tissuerejection; and/or (g) Universal stem cells by knocking out MHCreceptors—this approach would be used to generate cells of diminished oraltogether abolished immunological identity. Cell types for thisprocedure include but are not limited to, T-cells, B cells,hematopoietic stem cells, and embryonic stem cells. Therefore, thesestem cells or their derivatives (differentiated cell types or tissues)could be potentially engrafted into any person regardless of theirorigin or histocompatibility. (h) Targeted insertion of stem cell factorgenes at a safe-harbor locus (CCR5 locus or AAVS1 site located on humanchromosome PPP1R12c gene) within the human genome to reprogram cells toform induced pluripotent stem cells. (i) Targeted addition oftherapeutic genes at a safe-harbor locus (CCR5 locus or AAVS1 sitelocated on human chromosome PPP1R12c gene) within the human genome toprovide functional protein complementation in cells with correspondingdefective genes. (j) Targeted disruption of CCR5 by HDR or NHEJ toproduce HIV resistant cells. (k) Genetic engineering of humanpluripotent stem cells.

The compositions and methods can also be used for somatic cell therapy(e.g., autologus cell therapy and/or universal T-cell by knocking outMHC receptors, see section (g) above), thereby allowing production ofstocks of T-cells that have been modified to enhance their biologicalproperties. Such cells can be infused into a variety of patientsindependent of the donor source of the T-cells and theirhistocompatibility to the recipient.

In addition to therapeutic applications, the increased specificityprovided by the variants described herein when used in ZFNs or TALENscan be used for crop engineering, cell line engineering and theconstruction of disease models. The obligate heterodimer FokI cleavagedomain variant provide a straightforward means for improving ZFN andTALEN properties, especially when homodimer activity limits efficacy.

The engineered FokI cleavage domain variant described can also be usedin gene modification protocols requiring simultaneous cleavage atmultiple targets either to delete the intervening region or to alter twospecific loci at once. Cleavage at two targets would require cellularexpression of four ZFNs or TALENs, which would yield ten differentactive ZFN or TALEN combinations. For such applications, substitution ofour variants for the wild-type nuclease domain would eliminate theactivity of six of these combinations and reduce chances of off-targetcleavage.

In particular embodiments, a method is disclosed for cleaving genomiccellular chromatin in a region of interest, the method comprising: (a)selecting a first nucleotide sequence in the region of interest; (b)engineering a first zinc finger binding domain or transcriptionactivator-like effector (TALE) domain to bind to the first sequence; (c)expressing a first fusion protein in a cell, the first fusion proteincomprising the engineered zinc finger binding domain or transcriptionactivator-like effector (TALE) domain and an engineered FokI cleavagedomain variant; (d) expressing a second fusion protein in the cell, thesecond fusion protein comprising a second zinc finger binding domain ortranscription activator-like effector (TALE) domain and a second FokIcleavage domain variant; wherein the first fusion protein binds to thefirst nucleotide sequence, the second fusion protein binds to a secondnucleotide sequence from the first nucleotide sequence on thecomplementary strand of DNA, the first and second engineered cleavagedomains form a heterodimer that cleaves the cellular chromatin in theregion of interest.

In another embodiment, a method is disclosed for reducing the formationof FokI cleavage domain variant homodimers, comprising engineering andusing at least one FokI cleavage domain variant comprising a mutation inat least three or more wild-type amino acid residues to promoteheterodimer formation. The method may include minimizing cytotoxicityand/or eliminating or greatly reducing cellular toxicity, by reducingoff-target cleavage.

In yet another embodiment, a method for delivering a targeted genomicdouble-strand break (DSB) in cells is used, including plant, animal andhuman cells. The method may include: (a) selecting a first nucleotidesequence in the region of interest; (b) engineering a first zinc fingerbinding domain or transcription activator-like effector (TALE) domain tobind to the first sequence; (c) expressing a first fusion protein in acell, the first fusion protein comprising the engineered zinc fingerbinding domain or transcription activator-like effector (TALE) domainand an engineered FokI cleavage domain variant; (d) expressing a secondfusion protein in the cell, the second fusion protein comprising asecond zinc finger binding domain or transcription activator-likeeffector (TALE) domain and a second FokI cleavage domain variant;wherein the first fusion protein binds to the first nucleotide sequence,the second fusion protein binds to a second nucleotide sequence from thefirst nucleotide sequence on the complementary strand of DNA, the firstand second engineered cleavage domains form a heterodimer that cleavesthe cellular chromatin in the region of interest.

In certain aspects, human cells may include sensitive human primarycells, human embryonic stem cells (hESC), adult human stem cells, humanstem progenitor cells (hSPC) and human induced pluripotent stem cells(hiPSC). In yet other embodiments, method may further include deliveringeither stimulate local homologous recombination (HR) withinvestigator-provided donor DNA or inducing gene mutations at the siteof cleavage in the absence of a donor by non-homologous end joining(NHEJ) in cells.

In certain embodiments, a polynucleotide is disclosed encoding thepolypeptide of engineered FokI cleavage domain variant, e.g., FIGS. 1-6.In yet other aspects, an isolated cell or cell line are disclosedcomprising the polypeptide or the polynucleotide of the engineered FokIcleavage domain variant.

The invention is to be understood as not being limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

All publications mentioned herein, including patents, published patentapplications, and journal articles are incorporated herein by referencein their entireties including the references cited therein, which arealso incorporated herein by reference.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

This application claims priority to U.S. provisional application No.61/368,024, filed Jul. 27, 2010, which is hereby incorporated herein byreference in their entirety.

EXAMPLES Introduction

The creation of custom-designed zinc finger nucleases (ZFNs), and hencethe development of ZFN-mediated gene targeting, provides molecularbiologists with the ability to site-specifically and permanently modifyplant and mammalian genomes, including the human genome viahomology-directed repair of a targeted genomic DSB (1-5). The ZFNs areinactive as monomers. Mechanism of DSB by ZFNs requires that twodifferent ZFN monomers bind to their adjacent cognate sites on DNA andthat the FokI nuclease domains dimerize to form the active catalyticcenter for the induction of the DSB (6,7). Since dimerization of theFokI cleavage domain is required to produce a DSB, binding of two 3- or4-finger ZFN monomers (each recognizing a 9- or 12 bp inverted site) toadjacent sites is necessary for delivering a genomic DSB in cells. Sucha pair of ZFNs effectively has an 18- or 24-bp recognition site, whichis long enough to specify a unique genomic location in plant andmammalian cells, including human cells (8,9). Because the recognitionspecificities of the ZFPs can be easily manipulated experimentally,designer ZFNs offer a general way for targeted manipulation of thegenomes of a variety of cells and cell types (5, 14-31).

ZFN-mediated gene modification has been successfully demonstrated in avariety of cells from diverse species like frog oocytes (5), Drosophila(14-16), nematodes (17), zebra fish (18-20), mice (21), rats (22,23),plants (24,25) and humans (21, 26-31). High rate of endogenous genemodification efficiencies (>10%) have been achieved using this approach(27). However, in the case of ZFNs fused to wild-type FokI cleavagedomains (FokI_WT), homodimers may also form, which could limit theefficacy and safety of the ZFNs by inducing off-target cleavage (32-34).ZFNs toxicity resulting from off-target cleavage, particularly whenusing 3-finger ZFNs, has been reported to decrease the viability oftargeted cells. Two different approaches have been developed to reducethe cytotoxicity of ZFNs to cells: 1) Structure-based redesign of FokIcleavage domains at dimer interface to create obligate heterodimervariants that retained the wild type (WT) catalytic activity of naturalFokI enzyme, but show reduced off-target cleavage, which is discussedbelow (32-34); and 2) Attenuation of ZFN toxicity by small-moleculeregulation of protein levels in cells (35). The latter strategy,involves creating ZFNs with shortened half-lives by destabilizing ZFNseither by linking to a ubiquitin moiety to the N-terminus and thenregulating ZFN levels by using a small molecule proteosome inhibitor orlinking a modified destabilizing FKBP12 domain to the N-terminus andthen regulating ZFN levels by using a small molecule that blocksdestabilization effect of the N-terminal domain. Thus, it appears thatby regulating ZFN levels one could maintain high rates of ZFN-mediatedgene targeting while reducing ZFN toxicity.

Here, we report further improvements to obligate heterodimer variants ofFokI cleavage domain for creating designer ZFNs with minimal cellulartoxicity.

Example 1 Materials and Methods

Construction of ZFNs and the Donor Plasmid Substrate

The design and synthesis of CCR5-specific 3- and 4-finger ZFNs aredescribed in references 21 and 28. The obligate heterodimer variants ofFokI cleavage domain were constructed using overlapping oligonucleotidesas described in reference 21. The nucleotide and protein sequences ofthe various obligate heterodimer variant pairs of FokI cleavage domainare shown in Table 2. Construction of the mutant eGFP genes encoding thedesired ZFN target sites and the donor substrate for eGFP genecorrection are described in references 21 and 27. The protocol forgenerating HEK293 cell lines with an integrated mutant eGFP geneencoding the desired ZFN target sites are described in references 21 and27.

TABLE 2 Dimer Interface Interactions in Obligate Heterodimers of FokINuclease Domain Variants Amino Acid Redesign of Changes FokI Dimer orReplacements H-bond H-bond H-bond Hydrophobic Interface Chain ChainInteractions Interactions Interactions Interactions Residues (A) (B) (A)(B) (A) (B) (A) (B) (A) (B) FokI_WT 483-Asp 487-Arg R487-D483 D483-R487Q486-E490 I499-I538 (Aggarwal (D) (R) (Bi-dentate) (Bi-dentate) (SingleH- Lab)¹ 486-Gln 490-Glu (2.7/2.7 A°) (2.9/3.1 A°) bond) (Q) (E) 499-Ile499-Ile (I) (I) 538-Ile 538-Ile (I) (I) EL_KK 486-Glu 490-Lys R487-D483D483-R487 — — (Sangamo)² (E) (K) (Bi-dentate) (Bi-dentate) 499-Leu538-Lys (1.8/2.0 A°) (2.3/2.0 A°) (L) (K) RV_DA 483-Arg 487-AspR487-D483 R483-G480 Q486-D487 — (Cathomen (R) (D) (Bi-dentate) (SingleH- (Single H- Lab)³ 499-Val 538-Ala (2.7/2.7 A°) bond) bond) (3.8 (V)(A) (3.5 A°) A°) REL_DKK 483-Arg 487-Asp R487-D483 R483-G480 — —(PBPL/JHU)⁴ (R) (D) (Bi-dentate) (Single H- 486-Glu 490-Lys (2.7/2.7 A°)bond) (E) (K) (2.2 A°) 499-Leu 538-Lys (L) (K) RELV_DKAK 483-Arg 487-AspR487-D483 R483-G480 E486-K490 — (PBPL/JHU)⁴ (R) (D) (Bi-dentate) (SingleH- (Single H- 486-Glu 490-Lys (2.7/2.7 A°) bond) bond) (3.3 (E) (K) (3.5A°) A°) 499-Leu 499-Ala (L) (A) 538-Val 538-Lys (V) (K) ¹Wah et al(1998); ²Miller et al (2007); ³Szczepek et al (2007) and ⁴PBPL/JHU (thisexample)FACS and Microscopy Analyses

HEK293 cells carrying a mutated eGFP reporter gene were transientlytransfected with a donor plasmid carrying a fragment of wild-type GFPand plasmids expressing various 3- and 4-finger CCR5-specific ZFNconstructs using Lipofectamine 2000 are described in references 21 and27. Transfections of various obligate heterodimer variants and FokI_WTwere performed one after another on the same day. After eachtransfection, the treated cells were split into 2 flasks. GFP positivecells in about 10,000 treated cells in each flask were determined byFACS and then normalized to one million treated cells. The differencebetween the two independent FACS readings is shown as error bars. Three,five and seven days post-transfection with ZFNs and donor plasmid, eGFPgene correction was measured by FACS using a BD FACS Canto II. GFPfluorescence (GFP) was measured using BP 530/30 filter. BD FACS Diva™Software, v6.1.1 was used for analyses. GFP positive cells were sortedand examined by microscopy to confirm GFP expression. Independenttransfections, performed using 3-finger ZFN variant pairs on differentdays, showed a similar trend for gene correction efficiencies of EL_KKand REL_DKK, respectively.

Western Blot Analysis of Obligate Heterodimer Variants Expression inHEK293 Flp-In Cells

Western blot analysis was performed are described in reference 32.HEK293 Flp-In cells were grown and transfected are described inreference 21. After 30 hours cells were harvested and resuspended inRIPA buffer (Sigma-Aldrich). 50 μg of total protein of cell extract wereseparated by 10% SDS-PAGE and transferred to PVDF membrane (AmershamBiosciences). The blot was blocked and incubated with a rabbit anti-FokIantibody (1:200) followed by incubation with horseradishperoxidase-conjugated secondary antibody (Amersham Biosciences, 1:1000)and developed using an ECL chemiluminescence detection system (ThermoScientific) according to manufacture's instructions. The expressionlevels (band intensities) of Fok_WT (171.82), RV_DA (171.54), EL_KK(169.51) and REL_DKK (169.05), respectively, were quantified using theImage J software NIH, Version 1.36b).

Analysis of Genome-Wide Double-Strand Breaks in ZFN-Treated Cells

For cytotoxicity analysis, cells were transfected with 400 ng each ofZFN expression plasmid using Lipofactamine 2000 (Invitrogen) accordingto manufacture's instructions. Alternatively, cells were exposed to 10μM etoposide for 60 minutes 2 h before harvesting. Cells were collected30 h after transfection and fixed in ice-cold methanol for 15 min atroom temperature, permeabilized in 0.5% Triton X-100 and then blockedusing 5% serum for 45 minutes at room temperature. After blocking, cellswere then incubated with anti-53BP1 rabbit polyclonal antibodies (BethylLaboratories) overnight at 4° C. followed by incubation with AlexaFluor594-conjugated secondary antibodies (Invitrogen-Molecular Probes) with 2μg/ml of DAPI (Roche) at room temperature in the dark for one hour.Slides were mounted and analyzed by fluorescence microscope.

Results

Miller et al (2007) strategy for obligate heterodimer developmentcomprised of two key elements: First, ensure that the FokI nucleasedomain variants retained the desired catalytic activity by using thefunctional screen of GFP reporter gene correction in human cells (32).Second, use a stepwise approach to modification of the dimer interfaceusing rational design. This was done through four cycles of variantdesign and testing, each of which substituted one amino acid at thedimer interface providing incremental improvement in the specificity ofheterodimer formation. In each cycle of development, a small panel ofamino acid substitutions was generated within one cleavage domain, whileits partner was not modified. The choice of substitution was guided bythe co-ordinates of the native crystal structure of FokI restrictionendonuclease dimer and mutations were introduced at positions that couldcontact the unmodified partner with a bias towards charge-chargeinteractions. Each variant was screened for the ability to stimulategene correction in two different configurations, namely as a heterodimerwith the unmodified partner and as a homodimer, alternating successivedevelopment cycles between the two sides of the dimer interface. In eachcycle of development, they were able to identify a variant that wasefficiently induced gene correction as heterodimer, but showed reducedactivity as a homodimer. Through these stringent tests, Sangamo groupgenerated FokI cleavage domain pair with the double mutations, namelyQ486E:I499L (FIG. 10, SEQ ID NO: 19) and E490K:I538K (FIG. 11, SEQ IDNO: 21) (also known as EL_KK) that promote obligate heterodimerformation. EL_KK pair was shown to possess ˜10-fold reduced cytotoxicityas compared to FokI_WT cleavage domains.

Szczepek et al (2007) also used rational design based on the crystalstructure of the native FokI endonuclease and protein modeling toidentify critical residues involved in the dimerization of ZFNs (33).FokI endonuclease structure has shown that the dimerization is mainlymediated by helices α4 (residues 479-490 through a pair of salt bridgeformation between residues D483/R487) and α5 (residues 528-539) of thecleavage domain, which is also supported by functional data (36, 37).The model also predicted the existence of another contact between Q486and E490. Thus, the FokI crystal structure indicated that an asymmetricdimer interface could be created by rational redesign by swapping acritical pair of interacting residues, like D483R or R487D. Byincorporating similarly charged residues D483/D487 or R483/R487 in thesame FokI nuclease domain subunit, one could make formation homodimersunlikely, owing to electrostatic repulsion. Similarly, the residues Q486and E490 were redesigned to generate variants EE (Q486E) and QK (E490K)that favor heterodimerization over homodimerization. Cathomen labgenerated a FokI cleavage domain pair with the double mutations, namelyD483R:I538V (FIG. 9, SEQ ID NO: 17) and R487D:I499A (FIG. 8, SEQ ID NO:15) (also known as RV_DA) that promote obligate heterodimer formation.RV_DA pair was shown to possess reduced cytotoxicity as compared toFokI_WT cleavage domains. Later, DD_RR pair was also shown to havereduced cytotoxicity (34).

Engineering of Improved Obligate Heterodimer Variants of FokI CleavageDomain

The following example makes further improvements to the obligateheterodimer variants of FokI cleavage domain, to further minimizeoff-target cleavage and lower ZFN toxicity, and thereby increase theefficacy and efficiency of ZFN-mediated gene targeting of human cells.Such mutants also greatly increase the viability of gene-modified humancells, especially the sensitive human primary cells, human embryonicstem cells (hESC) and human induced pluripotent stem cells (hiPSC).Here, we used two different approaches to improve the obligateheterodimer variants of FokI cleavage domain for creating designed ZFNswith minimal toxicity.

We generated two pairs of new obligate heterodimer variants of FokIcleavage domain with lowered ZFN toxicity. The monomer of one of thesets contained the mutations, D483R:Q486E:I499L (FIG. 1, SEQ ID NO: 1),while its dimer partner contained the mutations, R487D:E490K:I538K (FIG.2, SEQ ID NO: 3). Together the pair is depicted as REL_DKK. The secondpair contained one more mutant residue in each of monomer chains inaddition to the above: D483R:Q486E:I499L:I538V (FIG. 3, SEQ ID NO: 5)and R487D:E490K:I499A:I538K (FIG. 4, SEQ ID NO: 7). Together the pair isdepicted as RELV_DKAK.

In a second approach, we replaced the FokI segment(IVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITN (SEQ ID NO: 23)) (see FIG. 12, SEQ ID NO: 23)containing the α4 and α5 helices (in italics and shaded yellow) thatmediate dimerization between two monomers of the FokI cleavage domainswith the corresponding segment from StsI, an isoschizomer of FokIendonuclease.

The segment in each of the monomer was replaced with LDSKAYSEGFPLTASHT RAMERYLRQFTERKEELKPTWWDIAPEHLDNTYFAYVSGSSFS GNYKEQLQKFRQDT (SEQ ID NO:68) (see FIG. 10, SEQ ID NO: 19) and ILDSKAYSEGFPLTASHTDAMG D YLKQFTERKEEIKPTWWDIAPEHLDNTYFAYVSGSFS GNYKEQLQKFRQ K T (SEQ ID NO: 69)(see FIG. 11, SEQ ID NO: 21) respectively, to further decrease theaffinity at the dimer interface. Furthermore, based on our results fromthe first approach, we also replaced three amino acid residues withinthe StsI segment of each monomer chain (which are shown in bold type andare double-underlined) to promote obligate heterodimer formation.Together the pair is referred to as FokI_StsI. Since many of the aminoacid residues of the StsI segment are quite distinct from the residueswithin the FokI segment, we reasoned that this would result in furtherdestabilization of the dimer interface and decrease the formation ofhomodimers, while promoting the heterodimers.

Protein modeling and energy minimization of the obligate heterodimervariant pairs of FokI cleavage domain, EL_KK, RV_DA, REL_DKK andRELV_DKAK respectively, were carried out using the SPDBV software(described in detail in the supplementary material). Visualization ofprotein structure rendering of images for H-bond interactions wereperformed with CCP4 MG software. The H-bond interactions that werepresent between the A and B monomer chains of the different obligateheterodimer variants are shown in FIG. 13. For the REL_DKK pair, themodel revealed only one bi-dentate H-bond between residues R487 of chainA and the D483 of chain B, predicting the weakest dimer interfaceinteractions between the two monomers.Testing the Efficacy and Efficiency of Engineered Obligate HeterodimerFokI Nuclease Domain Variants Using the Proxy GFP Gene TargetingReporter System in HEK293 Cells

We compared the efficiency and efficacy of gene targeting of the variouspairs of obligate heterodimer variants (REL_DKK; RELV_DKAK andFokI_StsI) with those of FokI_WT, EL_KK pair from Sangamo and RV_DA pairfrom Cathoman lab, by making fusions to the previously published pair of4-finger ZFPs that was shown to target CCR5 in human cells (28,29).ZFN-mediated gene correction at the mutant GFP locus was very efficientin HEK293 Flp-In cells, yielding GFP positive cells upon transductionwith the corresponding pairs of ZFNs containing either FokI_WT or theobligate heterodimer variants (FIG. 14). Transfection with donor alonedid not yield any GFP positive cells by microscopy. Quantitative FACSanalyses of the GFP positive cells at 3, 5 and 7 days post-transfectionwith designer various ZFN pairs and donor plasmids are shown in FIG.14A. The number of surviving GFP positive cells using the REL_DKK mutantpair at 3, 5 and 7 days post-transfection was consistently better ingene targeting experiments as compared to the other obligate heterodimervariant pairs, therefore that they are less toxic to cells, due toreduced off-target cleavage.

Once established, the gene-altered cells are viable and they continuedto increase in number for several weeks. We isolated 3 differentindividual GFP positive clones from gene targeting experiment REL_DKKmutant pair by serial dilution of FACS sorted GFP positive cells andgrew them for genotypic characterization. Three different loci, namelythe mutant GFP locus, the endogenous CCR5 locus and the CCR2 locus, werePCR-amplified using the corresponding locus-specific primers from theisolated genomic DNA of the individual GFP positive clones. The PCR DNAfrom the mutant GFP locus of the gene-altered clones were all resistantto BstXI digestion indicating that gene correction to the wild-typesequence has occurred in the GFP positive clones. The PCR DNA from thethree different loci was then cloned in the pGEMT vector fortransformation into E. coli. DNA sequence analysis of at least 4recombinant E. coli clones from each individual GFP positive cloneconfirmed that gene correction indeed has occurred. DNA sequenceanalysis of 4 recombinant bacterial clones generated by cloning thePCR-amplified DNA of the endogenous CCR5 locus from each of the threeindividual GFP positive clones, as expected, showed simple deletionand/or insertion mutations at the targeted 4-finger CCR5 site resultingfrom NHEJ (FIG. 15). Extensive genotypic characterization of the GFPpositive cells from gene targeting experiments using 4-fingerCCR5-specific ZFNs is described in reference 21.

We then compared the efficiency and efficacy of gene targeting ofREL_DKK obligate heterodimer variant pair with those of FokI_WT andEL_KK pair from Sangamo by making fusions to the 3-finger ZFPs thattarget CCR5 in human cells (13). Unlike the CCR54-finger ZFNs which hadno linker, the active CCR5-specific 3-finger ZFNs contained the (Gly₄S)₃linker between the ZFPs and the FokI nuclease domain variants, sincethey are designed to target ZFN sites separated by a 12 bp spacer (13).Transfection with donor alone or ZFPs fused to FokI_WT and donor did notyield any GFP positive cells by microscopy (FIG. 14B). ZFN-mediated genecorrection at the mutant GFP locus was very efficient in HEK293 Flp-Incells, upon transfection with 3-finger ZFPs fused to REL_DKK or EL_KKobligate heterodimer FokI nuclease domain mutants and donor, yieldingGFP positive cells (FIG. 14B). Quantitative FACS analyses of the GFPpositive cells at 3, 5 and 7 days post-transfection are shown in FIG.14C. The GFP positive cells using the REL_DKK mutant pair at 3, 5 and 7days post-transfection was consistently higher in gene targetingexperiments as compared to EL_KK pair, suggesting that REL_DKK variantsare less toxic to cells, which is likely due to further reduction inoff-target cleavage. Results from two independent transfections,performed on different days, are shown in FIG. 16; both transfectionsshowed a similar trend for gene correction efficiencies of EL_KK andREL_DKK, respectively. We also performed titrations of 3-finger ZFNexpression plasmids of obligate heterodimer variants EL_KK and REL_DKKat 0.2, 0.4 and 0.8 μg respectively, with constant donor plasmid (1.0μg) to obtain a dose response curve to study the differences between thetwo mutants (FIG. 17). REL_DKK variant consistently yielded more genecorrected cells as compared to EL_KK mutant. The maximal difference ofGFP corrected cells was observed 5 days post co-transfection of plasmids(FIG. 17).

Once established, the gene-corrected cells are viable and they continuedto increase in number for several weeks. We isolated 9 differentindividual GFP positive clones from gene targeting experiment usingREL_DKK mutant pair by serial dilution of FACS sorted GFP positive cellsand grew them for genotypic characterization. Three different loci,namely the mutant GFP locus, the endogenous CCR5 locus and the CCR2locus, were PCR-amplified using the corresponding locus-specific primersfrom the isolated genomic DNA of the individual GFP positive clones. ThePCR DNA from the mutant GFP locus of the gene-altered clones were allresistant to BstXI digestion indicating that gene correction to thewild-type sequence has occurred in the GFP positive clones (FIG. 14D).The PCR DNA from the three different loci was then cloned in the pGEMTvector for transformation into E. coli. DNA sequence analysis of atleast 4 recombinant E. coli clones from each individual GFP positiveclone confirmed that gene correction indeed has occurred. DNA sequenceanalysis of 4 recombinant bacterial clones generated by cloning thePCR-amplified DNA of the endogenous CCR5 locus from each of theindividual GFP positive clones, as expected, showed simple deletionand/or insertion mutations at the targeted 3-finger CCR5 site resultingfrom NHEJ (FIG. 15). No change in the nucleotide sequence of the CCR2locus was observed in the limited number of GFP positive clones thatwere sequenced suggesting that the designed pair of 3-finger CCR5ZFNsdid not cleave at a distantly related site (data not shown).

Reduced Levels of DNA Damage by REL_DKK Heterodimer Variant Pair

As a direct measurement for ZFNs' cytotoxicity, we then monitoredwhether REL_DKK heterodimer variant pair reduced genome-wide DNAcleavage levels when expressed in human HEK293 Flp-In cells using thewell-established assay for visualizing DNA double-strand breaks asdescribed in reference 32. We used antibody-mediated detection of theprotein 53BP1, which localizes to sites of DNA damage and forms focithat are visualized by immunofluoroscence. Antibody-based detection of53BP1 revealed that substitution of either EL_KK or REL_KK heterodimervariant pair for FokI_WT cleavage domain showed marked reduction in thenumber of 53BP1-stained foci in HEK293 Flp-In cells (FIG. 18A).Moreover, the fraction of 53BP1-stained cells with multiple foci (>3foci) for REL_DKK heterodimer variants was lower than that of EL-KKvariants (FIG. 18A). We confirmed that the observed results were not dueto poor protein expressions of REL_DKK variants in HEK293 Flp-In cells,since western blot analysis showed comparable levels of ZFN expressionsfor FokI_WT, EL_KK and REL_DKK, respectively (FIG. 18B).

Discussion

This example details further improvements to obligate heterodimervariants, by incorporating multiple mutations at the dimer interface ofFokI cleavage domains, to eliminate or greatly reduce ZFN toxicity,while increasing the efficacy and efficiency of ZFN-mediated genetargeting in cells. Our results indicate that the REL-DKK pair fused to3- and 4-finger ZFPs consistently performed better as compared to EL_KKor RV_DA, suggesting this pair exhibits the lowest toxicity to humancells, while retaining catalytic efficiency of the FokI_WT cleavagedomains.

Using 3D protein modeling based on FokI crystal structure and energyminimization calculations, we analyzed the H-bond and hydrophobicinteractions present at the dimer interface of the newly generatedmutant pairs. The RELV_DKAK model predicts the existence of oneadditional H-bond interaction including those that are present inREL_DKK pair, which likely provides added stability to the dimerinterface. The FokI_StsI pair generated by replacing the FokI segment(encoding the α4 and α5 helices that are involved in the dimer interfaceinteractions) with the StsI segment in FokI nuclease domain, althoughactive, appears to have much diminished catalytic activity, which isprobably due to the destabilizing effect of the StsI segment on proteinfolding of the FokI nuclease domains.

While the 4-finger CCR5ZFNs (created by fusing highly specific 4-fingerZFPs to FokI_WT cleavage domains) were efficient in GFP gene correctionassays, the 3-finger CCR5-specific ZFNs (created by fusing modular3-finger ZFPs to FokI_WT cleavage domains) did not yield any GFPpositive cells, suggesting that sequence-specificity of the designedZFNs is the major determinant of ZFN activity for efficient genetargeting and for greatly reduced cellular toxicity. The toxicityresulting from off-target cleavage could be attributed to: (i) Homodimerformation by the individual ZFN species; and (ii) Relaxed specificity ofthe ZF modules (used to generate ZFPs by modular assembly), whichresults in degenerate or off-target binding by ZFNs (10). However, whenthe corresponding CCR5-specific ZFPs were fused to the obligateheterodimer variants of FokI nuclease domains, the ZFNs were active inthe GFP gene targeting reporter system indicating that the off-targetcleavage could be eliminated or greatly reduced by fusing 3-finger ZFPsto obligate heterodimer variant pair (REL_DKK).

Our results from GFP gene correction experiments are consistent with themechanism of double-strand cleavage by natural FokI enzyme (36-39) andby ZFNs (7). Bitinaite et al (1998) reported that although FokI enzymebinds DNA as a monomer, dimerization of the nuclease domains is requiredto form active sites in order to cleave DNA (36, 37). Later studies haveshown that an active dimer could form with just one subunit bound to itsrecognition site, albeit through a weak protein-protein interaction ofthe nuclease domains, particularly at high enzyme concentrations (38).Studies on the mechanism of cleavage by ZFNs suggest that binding of twoZFN monomers to two binding sites is required for effectivedouble-strand DNA cleavage (7). We speculate that ZFN monomers bound toa single site are unlikely to form an active dimer by associating withanother ZFN monomer that is not bound to DNA, since this has to occurthrough the weak protein-protein interaction. It is more likely that theoff-target cleavage results, when one ZFN monomer is bound to itscognate site while the other is bound to a nearby degenerate site (sinceall ZF modules do not always contact all the three bases within theircognate triplets, which could result in relaxed specificities) or boundnon-specifically to DNA, especially at high protein concentrations.Interactions at the dimer interface could provide additional stabilityto the off-target cleavage site complexes, inducing DSB at these sites,resulting in ZFNs' toxicity to human cells. In such instances,destabilization of the dimer interface would greatly diminish ZFNsoff-target cleavage, leading to lowered toxicity to cells. Our resultslend support to this idea.

ZFN technology applications in human therapeutics depend on the abilityto create custom-designed ZFNs that cleave the target sequences withexquisite sequence-specificity and high affinity (27). Highly specificZFPs fused to the re-engineered obligate heterodimer variant pair (likeREL_DKK described in this example) will be critical for eliminating orgreatly reducing ZFN toxicity to human cells. This will allow one todeliver a targeted genomic DSB to human cells, while leaving the rest ofthe genome unchanged. Such engineered highly specific ZFNs will enablewider application of ZFN technology in human therapeutics, by furtherincreasing the viability of the gene-modified cells, especially thesensitive human primary cells, human embryonic stem cells (hESC) andhuman induced pluripotent stem cells (hiPSC).

References cited herein are listed below for convenience and are herebyincorporated by reference in their entirety.

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We claim:
 1. A polypeptide comprising either a first engineered FokIcleavage domain variant D483R:Q486E:I499L (SEQ ID NO: 1), or a secondengineered FokI cleavage domain variant R487D:E490K:I538K (SEQ ID NO:3), the first engineered FokI cleavage domain variant forming anobligate heterodimer with the second engineered FokI cleavage domainvariant.
 2. The polypeptide of claim 1, wherein the obligate heterodimercomprises a first monomer containing the polypeptide designatedD483R:Q486E:I499L (SEQ ID NO: 1), and a second monomer containing thepolypeptide designated R487D:E490K:I538K (SEQ ID NO: 3).
 3. Thepolypeptide of claim 1, further comprising a DNA-binding domain.
 4. Thepolypeptide of claim 3, wherein the DNA-binding domain comprises zincfinger protein (ZFP) domain or transcription activator-like effector(TALE) domain.
 5. A polynucleotide encoding the polypeptide of theclaim
 1. 6. An isolated cell or cell line comprising the polypeptide ora polynucleotide encoding the polypeptide of the claim 1.